TETRADECAMERIC CHAPERONIN-60 CAN BE ASSEMBLED IN-VITRO FROM MONOMERS IN A PROCESS THAT IS ATP INDEPENDENT

The present work shows that monomers of cpn60 (groEL) formed at 2.5 M urea could be assembled to tetradecamers in a process that was independent of ATP. Reassembled cpn60 was able to assist the folding of urea unfolded rhodanese. When cpn60 was incubated at urea concentrations higher than 2.75 M, assembly of tetradecameric cpn60 did not occur after dialysis, and the presence of ATP did not stimulate the assembly process. The cpn60 used here did not display the previously reported ATP-dependent self-assembly of cpn60 monomers that required a higher urea concentration (4 M) for formation (Lissen et al. (1990) Nature 348, 339-342). Assembly and disassembly of cpn60 tetradecamers were followed as a function of the urea concentration by ultracentrifugation and gel electrophoresis in the presence of urea. The electrophoresis results demonstrate that there is rapid assembly of tetradecamers following preincubation and rapid removal of urea at concentrations lower than 2.5 M. Thus, previous methods monitored irreversible dissociation of cpn60, and the present results indicate that the cpn60 assembly requirements for ATP are dependent on pretreatment conditions.